Transparent Biology
By Shan Zhao
Peering into intact human organs with single-cell resolution
Peering into intact human organs with single-cell resolution
Five centuries after the pioneering anatomical drawings of Andreas Vesalius, medical researchers remain hampered by their limited ability to view the inner workings of the human body.
With imaging of sufficient clarity, doctors could observe early signs of cancer metastasis, for instance, or they could detect aneurysms and other small changes that indicate major health risks. They could study the workings of the brain’s vasculature, or understand the construction of the kidneys at a cellular level. Such information could even make it possible to grow replacement organs for patients—a technique that is feasible in principle, if only researchers had an accurate blueprint to work from. But until recently, the requisite imaging did not exist.
Even the best views of living tissue obtainable by magnetic resonance imaging (MRI) and computerized tomography (CT) result in only millimeter-scale resolution. That level of precision is remarkable in many ways—good enough to assist disease diagnosis or to map out the overall flow of blood through the brain, for example—but it is far too crude for more advanced tasks. Mapping the body’s cellular structure would require micrometer-scale resolution.
Courtesy of Shan Zhao
To examine the detailed morphologies of organs, researchers often turn instead to traditional histology. That approach requires cutting organs into thin, serial sections and immersing them in dye solutions to highlight structural patterns in the tissue. Then the researchers must image every slice and use software to reconstruct the whole organ from those thousands of images. In 2013, Katrin Amunts and her colleagues at the Institute of Neuroscience and Medicine in Jülich, Germany, published an ultrahigh-resolution three-dimensional model of the human brain, dubbed BigBrain, the largest example of this type of work. Over several years, this team reassembled 7,404 slices from the brain of a 65-year-old. Such studies require tremendous labor and still yield far-from-ideal results; slicing the organ alters and damages the tissue, and sections can be lost.
My colleagues and I at Ludvig Maximilian University of Munich sought a better way. We wanted to create higher-resolution 3D maps of intact human organs, and to do so without damaging the tissue. After two years of effort, we have succeeded. We have developed a scalable, robust process that renders intact human organs transparent using chemical cocktails. Then we can image the organs directly using a technique called light-sheet microscopy. In this way, we are seeing the body as never before, examining eyes, thyroids, kidneys, and other structures at the single-cell level (see video below).
A key problem in observing whole organs, or any large piece of tissue, is that light can penetrate only to depths of no more than hundreds of micrometers. Tissues include components such as water, lipids, proteins, and carbohydrates, each with its own refractive index (a measure of how fast light travels through a material compared with its speed through a vacuum—higher values represent lower speeds). These components are distributed throughout the organs in different structural patterns to form cells, blood vessel networks, connective fibers, and a complex mesh of extracellular matrix. When light strikes these components, photons scatter and are absorbed.
In principle, that problem could be overcome by equalizing the optical properties of all the different components, but that task would be highly complicated. Water has a refractive index of 1.33 and composes up to approximately 80 percent of tissue. Proteins and lipids each make up about 10 percent of tissue and have refractive indices of 1.50 and 1.48, respectively. With age, human organs accumulate other obscuring molecules, such as lipofuscin, melanin pigments, hemoglobin, and insoluble collagen, all of which can absorb light and make imaging adult tissue even more difficult.
When light strikes tissue components, photons scatter and are absorbed.
The idea of making tissues transparent by manipulating refraction originated more than 100 years ago. In 1914, German anatomist Werner Spalteholz at the University of Leipzig created the first transparent human anatomical specimens. He embedded tissue samples into media of different refractive indices and discovered that light would pass through when the medium’s refractive index matched that of the tissue. (This scientific idea also formed the basis for invisibility in H. G. Wells’s 1897 science fiction classic, The Invisible Man.) Other scientists tried to modify and improve Spalteholz’s method to avoid tissue necrosis, bubble formation, and blurring of structural details, but with only modest success.
Starting about a decade ago, several laboratories revisited the idea of creating transparent tissue, this time using a process called tissue clearing. This approach uses chemical cocktails to eliminate light scattering from lipid molecules and to remove light-absorbing molecules such as heme (a component of hemoglobin). Then researchers immerse the remaining tissue components in a solution that matches the remaining tissue’s refractive index.
Three kinds of tissue clearing technologies have been developed to systematically study animal and human specimens: hydrophobic (water-repellent) solvent-based clearing, hydrophilic (water-attractive) reagent-based clearing, and hydrogel-based clearing. Hydrophobic clearing systems have been the most effective in clearing larger animal samples. In 2016, Ruiyao Cai and her colleagues from the Institute for Stroke and Dementia Research in Munich reported turning an adult mouse transparent; three years later, they described its full neuronal connectivity. These tissue clearing methods don’t work for whole human organs, since they are limited to sections no more than 8 millimeters thick. Nevertheless, they proved inspirational.
In 2017 we decided to examine tissue clearing systematically and to explore whether we could use hydrophobic solvents to clear larger, thicker pieces of human tissue so that we could map intact human organs at cellular resolution. We started by working to improve the existing tissue-clearing methods. We swapped chemicals, adjusted concentration or incubation times, and tried different reagents as we endeavored to clear and match the refractive indices within the tissue. After several months of work, however, none of these combinations gave us transparent organs.
At this point, we regrouped and examined why human tissues are so much more difficult to make transparent than rodent tissues, which can be cleared with more than 40 different chemical cocktails. The organs from adult humans are much larger and sturdier than those from rodents that have lived just a few months. Human tissue structures also include myriad hydrophilic and hydrophobic components that are intertwined to form thousands of layers of mesh. The fatty, lipid molecules include diverse mixtures, and nerve structures are coated with the protein myelin. Whether we tried water-soluble or lipid-soluble solvents, we couldn’t get the chemicals to penetrate deeply within human tissue, so the resulting clearing was inefficient. We needed to add a different chemical—some special ingredient capable of permeating the intact organ to make it accessible to other kinds of clearing chemicals.
Detergents looked like the best candidates, because these molecules have one portion that interacts with greasy tissue components and another that can interact with the tissue’s water. In a water solution, the greasy ends of detergents tend to clump together, pointing their water-loving ends out to form spherical structures called micelles. We tried sodium dodecyl sulfate and nonionic Triton X-100, two common tissue detergents, but we discovered that their micelles are too large. They remained on the tissue surface rather than penetrating into the dense mesh of human organs. We needed a special detergent that could form smaller micelles.
After an extensive search, we found our special ingredient. It has a long technical name, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate hydrate, commonly shortened to CHAPS. It is a commercially available detergent, but one that had never before been applied in this way. CHAPS forms micelles approximately one-fourth the size of those created by the other detergents.
Using CHAPS, we could finally make whole human organs fully accessible to the other tissue-clearing solvents. We experimented with many chemicals to develop our tissue-clearing process: hydrophilic animoalcohols for removing color from heme, acids for loosening the extracellular matrix, miscible alcohols to replace the tissue’s water, hydrophobic solvents for extracting the lipids, fluorescent dyes for labeling specific structures, and, in the end, a refractive index matching solution for full transparency. Our full recipe for these chemical cocktails has its own mouthful of a name: We call it small-micelle-mediated human organ efficient clearing and labeling, or SHANEL.
In the SHANEL process, we use a commercial pump to perfuse the chemical cocktail through an organ’s vascular system. The tissue-clearing process requires patience: A human kidney can be cleared within six weeks, but a larger, intact human brain takes approximately four months. After clearing, we apply fluorescent dyes to label and highlight cellular structures. Then comes the truly exciting part.
With these cleared and labeled organs, we can image the inner structures of intact human organs with a microscope. Working with LaVision BioTec GmbH, a German bioimaging company, we have developed a custom version of a light-sheet microscope. This device works by illuminating the sample with a beam of light that is just several microns thick, effectively “cutting” the tissue into sections using only light. Our modified light-sheet fluorescent microscope is the only one in the world that can hold samples as large as a human kidney.
A human kidney can be cleared within six weeks, but an intact human brain takes approximately four months.
As the microscope illuminates each tissue slice from both sides, the light activates the fluorescent molecules within the cleared organ and collects their signals. The sample holder moves the sample in steps, nudging it a few micrometers each time, so that every plane of tissue within a whole mounted organ is recorded. This approach mirrors the methods of traditional histology, allowing us to examine an organ layer by layer but without any destructive cutting.
We complete our imaging process by digitally combining these thousands of sequential images to observe the cellular details of intact human organs. Even allowing for the time needed to turn the organ transparent, SHANEL ends up being much faster than traditional histology. It is more accurate as well. To work with the extraordinary amount of high-resolution imaging data—terabytes of raw data alone—we needed powerful computers and a fast, accurate artificial intelligence algorithm to analyze our results. We have assembled the first detailed 3D maps of the intact human eye and kidney, and we have worked with slices of the human brain 1.5 centimeters thick. We can run the SHANEL clearing process on several organs simultaneously using a four-channel pump. The process is robust and could be easily implemented in other laboratories. Moreover, the transparent organs are hard and stable, allowing them to be stored long-term for future research. Right now, we’re limited by the size and power of our microscopes, not by the ability to clear tissues.
Using SHANEL, we can create reference maps of human organs with single-cell resolution, a digital anatomy text for the 21st century. With such maps of healthy organs, we can study the morphological changes that occur as a result of diseases, drugs, or other environmental factors. For example, we are interested in imaging organs from victims of coronavirus disease 2019 (COVID-19) to study the SARS-CoV-2 virus’s effects on tissue structures.
Complete maps of human organs and their functional details could eventually help researchers produce 3D, bioprinted organs that imitate natural tissue. Several teams have already begun developing functional lab-grown kidneys. For such organs to be transplanted into patients, though, they must be mechanically stable and must contain vascular and tubular networks that feed cells and collect urine as effectively as their natural versions. A bioprinted kidney must also include a glomerulus, a network of small blood capillaries encapsulated within a filtering unit. SHANEL imaging can provide the necessary structural guidance. We are currently constructing our own bioprinter and are studying the necessary vascular and stem-cell biology so that we can eventually build functioning organs in the laboratory.
Courtesy of Shan Zhao
We also continue to refine our organ-clearing technology. We are optimizing the process so that we can render organs transparent within several days instead of several weeks, and can take advantage of the autofluorescence of human tissues, which would allow us to skip the fluorescent labeling step. We are building a larger, more powerful light-sheet microscope that will be able to handle whole human brains. More detailed imaging data will also require more computing power and improved artificial intelligence algorithms.
This project has involved chemists, biologists, engineers, and computer scientists working together to discover the long-hidden details lurking deep within human organs. As we work on a technology that is both useful and beautiful, we hope that the ability to image whole organs in depth will inspire new insights and scientific creativity.
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